A novel limonin derivate modulates inflammatory response by suppressing the TLR4/NF-κB signalling pathway.

In our previous studies, we have demonstrated that a novel water-soluble derivative of limonin, (12S,12aS,Z)-8-((2-(diethylamino)ethoxy)imino)-12-(furan-3-yl)-6,6,8a,12a-tetramethyldodecahydro-1H,3H-oxireno[2,3-d]pyrano[4',3':3,3a]isobenzofuro[5,4-f]isochromene-3,10(9aH)-dione (V-A-4), exhibited strong anti-inflammatory activity both in vitro and in vivo. The purpose of this study was to further explore the underlying mechanisms of such activity demonstrated by V-A-4. The protective effect of V-A-4 on the alleviation of xylene-induced ear swelling and carrageenan-induced subcutaneous air pouch model was detected in vivo. Furthermore, the in vitro effects of V-A-4 and its mechanisms of action were determined by colorimetric COX (ovine) inhibitor-screening assay and in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This study showed that V-A-4 does not exert anti-inflammatory effect through the inhibition of COX-1 or COX-2. Rather, it is exerted through the suppression of the secretion of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), as well as through the infiltration of inflammatory cells. V-A-4 demonstrated strong inhibition of NF-κB activation through repression of IKKα and IKKß phosphorylations, which in turn leads to the phosphorylation and degradation of IκBα in LPS-induced RAW264.7 cells. Moreover, toll-like receptor 4 (TLR4) pathway was involved in the anti-inflammatory effect of V-A-4, which also played an important role in the down-regulation of LPS-mediated miR-146a and miR-155 expressions. These results encourage further development of V-A-4 as a potential candidate for the treatment of inflammatory diseases.

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model.

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system.